Figures

Abstract

The mechanism controlling the exit from herpes simplex virus latency (HSV) is of
central importance to recurrent disease and transmission of infection, yet
interactions between host and viral functions that govern this process remain
unclear. The cascade of HSV gene transcription is initiated by the
multifunctional virion protein VP16, which is expressed late in the viral
replication cycle. Currently, it is widely accepted that VP16 transactivating
function is not involved in the exit from latency. Utilizing the mouse ocular
model of HSV pathogenesis together with genetically engineered viral mutants and
assays to quantify latency and the exit from latency at the single neuron level,
we show that in vivo (i) the VP16 promoter confers distinct regulation critical
for viral replication in the trigeminal ganglion (TG) during the acute phase of
infection and (ii) the transactivation function of VP16 (VP16TF) is uniquely
required for the exit from latency. TG neurons latently infected with the VP16TF
mutant in1814 do not express detectable viral proteins following stress, whereas
viruses with mutations in the other major viral transcription regulators ICP0
and ICP4 do exit the latent state. Analysis of a VP16 promoter/reporter mutant
in the background of in1814 demonstrates that the VP16 promoter is activated in
latently infected neurons following stress in the absence of other viral
proteins. These findings support the novel hypothesis that de novo expression of
VP16 regulates entry into the lytic program in neurons at all phases of the
viral life cycle. HSV reactivation from latency conforms to a model in which
stochastic derepression of the VP16 promoter and expression of VP16 initiates
entry into the lytic cycle.

Author Summary

Herpes simplex virus (HSV) establishes life-long latent infections in sensory
neurons of the human host. Periodically, HSV exits latency in an infected neuron
and is transported to the body surface where it replicates, leading to recurrent
disease and infection of new hosts. We do not currently understand how entry
into the lytic cycle is blocked in neurons and latency is established. Nor do we
know how, at some time in the future, the lytic program becomes activated in the
one or two latently infected neurons which characterize a reactivation event. In
tissue culture cells, and by analogy in cells at the body surface, the HSV
replication program is initiated by the interaction of a virion protein, VP16
(brought in with the virus as a protein), with host cell factors. Here we show
that the de novo synthesis of VP16 is required for efficient viral replication
during the acute phase of infection in neurons. This indicates that latency is
favored because VP16 may not be transported efficiently to the nerve cell
nucleus. Once latency is established, the de novo expression of VP16 is an
absolute and very early requirement for the exit from the latent state. Our data
support a model of HSV reactivation in which the stochastic derepression of the
VP16 promoter and resulting expression of VP16 starts the viral lytic
program.

Funding: This work was funded by Public Health Service grants R01 AI 32121 (NMS) and R01
EY 13168 (RLT). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Primary infection with herpes simplex virus (HSV), universally the result of close
contact with an infected individual, is accompanied by dissemination of viral
genomes into the host nervous system. Although symptoms of the primary infection
usually resolve, large numbers of viral genomes remain in a transcriptionally
repressed state within neurons of sensory ganglia and the brain for the life of the
infected individual [1]. Periodically, stimulated by various stressors,
latency is exited and infectious virions are generated in a small number
(<0.05%) of latently infected neurons [2]–[5] which
transport virus back to the body surface through innervating axons. Although
individual neurons supporting lytic viral replication do not survive this process
[4]–[6], the large reservoir of
latently infected neurons allows this cycle to occur repeatedly which is the
mechanism of transmission and the cause of serious sequellae including blindness and
encephalitis. That 70–90% of the human population worldwide is
now infected is a testament to the efficacy of this strategy [7]. There is currently no
way to either eliminate latent virus or to prevent the exit from latency and no
effective vaccine to protect the uninfected, thus transmission rates remain high. To
date, the molecular mechanisms regulating reactivation from latency remain unclear.
Identifying the interactions between the neuron and latent viral genome that result
in the exit from latency is critical toward progress in understanding and ultimately
controlling this complex process.

In a striking case of parallel evolution, most DNA viruses employ strong enhancers to
promote the transcription of the earliest viral genes [8]. HSV differs from other
DNA viruses including most other herpesviruses in that transcription of its
immediate early (IE) genes is principally dependent on a protein component of the
virion that is a potent transcriptional activator [9],[10]. This
multifunctional late gene protein, VP16 (VMW65, α-TIF, UL48), interacts with
host cell proteins including HCF-1, a cell cycle regulator, and Oct-1, a POU domain
transcription factor, to form the VP16 induced complex (VIC) that binds to TAATGARAT
elements present in the five HSV-1 immediate early gene promoters [11]–[14]. Considering the complex
in vivo life cycle of HSV, the dependence on a structural protein produced late in
the infectious cycle to initiate transcription from the viral genome presents a
conundrum. How can the latent viral genome initiate the transcription of lytic phase
genes in the absence of this crucial transcriptional activator? Studies in the early
1990's led to the dogma that VP16 is simply not involved in reactivation
[15]–[17] and that its
function in initiating the lytic cycle is fulfilled by another viral function or a
host cell factor.

There have been two long-standing hypotheses regarding the initiation of the lytic
cycle during reactivation from latency. The first hypothesis proposes that the viral
IE gene ICP0 initiates reactivation from latency [18]–[21]. The
second proposes that viral early gene expression and DNA replication precedes and is
required for efficient IE gene expression during reactivation from latency [22],[23]. In these studies, reactivation was evaluated
using axotomized and explanted ganglia. Although this assay has been widely
utilized, it has become increasingly clear that explant reactivation does not model
HSV reactivation as it occurs in vivo [24],[25]. In hindsight, this
is not a surprising finding, in that axotomized and explanted neurons rapidly
undergo radical transcriptional changes, including apoptosis [24],[26],[27]. Recent reexamination
of these hypotheses using in vivo reactivation and single neuron level approaches
have demonstrated that the exit from latency does not require either a viral DNA
pre-amplification step [28] or functional ICP0 [25]. An important clue as
to how exit from latency is regulated came from the analysis of a viral mutant
termed ΔTfi in which a 350 bp region of the ICP0 promoter, which includes
the TAATAGARAT element through which VP16 transactivates this IE gene, is deleted.
Although this mutant reactivates with wild type kinetics in explant assays [25],[29], in
vivo it is severely impaired in its ability to reactivate, suggesting that
transactivation by VP16 may indeed be critical in the regulation of reactivation in
vivo [25].

Here we report results from experiments designed to test the hypothesis that VP16
regulates the exit from latency. Our studies support the hypothesis that in elegant
simplicity, the major coordinator of IE gene expression and tegument protein, VP16,
functions to regulate entry into the lytic program at all phases of the viral life
cycle. We find that in vivo (i) the VP16 promoter confers distinct regulation
critical for viral replication in the trigeminal ganglion, and (ii) VP16
transactivating function is required for reactivation from latency. Importantly,
that VP16 transactivation function (VP16TF) is required very early in the exit from
latency is supported by (i) failure of latent viral genomes to enter the lytic cycle
(as defined by expression of lytic viral protein) uniquely in the absence of VP16TF
(ICP0 null, viral thymidine kinase null, and tsICP4 mutants do exit latency), and
(ii) the restoration of reactivation competency of ΔTfi by replacement of
the TAATGARAT element. In the nervous system, de novo expression of VP16 from the
latent viral genome allows VP16 to coordinate the expression of the viral IE genes
and thereby initiate the productive lytic cycle.

Results

The VP16 promoter has unique regulatory properties in neurons in vivo

HSV initiates the viral lytic cycle under two distinct conditions, (i) following
infection of a cell by the virion, and (ii) from the latent viral genome. In the
first case, the lytic cycle is engaged through coordinated activation of the
viral IE genes by the virion associated transactivator, VP16 [11]–[14]. How the lytic cycle
is initiated from the latent genome remains unknown, although it is reasoned
that VP16, expressed with late kinetics during the lytic cycle, does
not supply this function [15], [16],
[18],
[30]–[33].

ICP0 null mutants can exit latency (demonstrated by the detection of lytic viral
protein expression), however, progression to lytic virus production
(reactivation) does not occur [25]. In addition, a mutant in which the VP16
binding site has been deleted from the ICP0 promoter also fails to reactivate in
vivo. Together, these findings raise the possibility that VP16 may play an
unexpected role in the regulation of IE genes very early in the exit from
latency. If this were the case, the regulation of VP16 must be distinct in this
context, with the protein expressed as a very early event and not as a standard
leaky late gene. To test this, we asked whether another viral promoter of
equivalent strength and kinetic class [34]–[36] could
confer “proper” regulation of VP16 in vivo. The VP5 promoter
was selected since replacement of this viral promoter with that of VP16 has been
reported previously and no measurable effect on the ability of the virus to
replicate in vivo, either at the surface or in the nervous system was observed
[37]. Thus the converse mutant in which the VP16
promoter/5′utr was replaced with that of VP5 was generated as detailed
in methods. A diagram of this mutant is
shown in Figure 1A. Three
independently derived viral mutants were characterized in vitro and in vivo.
Levels of VP16 mRNA in rabbit skin cells (RSC) infected with mutant VP5p/VP16
were not reduced compared to 17syn+ as quantified by northern blot
analysis at 6, 8 and 12 hr pi (not shown). Standard single (not shown) and
multi-step replication kinetic analysis in RSC revealed no alterations when
compared to the parental strain 17syn+ or the genomically restored
mutant VP5p/VP16-1R (Figure
1B). In order to determine the effect, if any, of this promoter exchange
on viral replication in vivo, five groups of 16 mice each were inoculated on
scarified corneas with 1×105 pfu of either VP5p/VP16-1,-3,
-5, VP5p/VP16-1R, or 17syn+. Titers of infectious virus in the eyes and
trigeminal ganglia (TG) were determined independently in three mice from each
group on days 2, 4, 6, 8, and 10 pi. The total amount of infectious virus
detected in the eyes during the acute stage of infection was not different among
the viruses compared, (area under the curve
(AUC) = 292, 261 vs. 250,013 or 281, 982,
respectively) (Figure 1C).
Note also that the peak viral replication occurring in the eyes on day 4 pi was
not different (p = 0.65; ANOVA ). In contrast,
total infectious virus detected during the acute stage in the TG was more than
200 fold reduced for the VP5p/VP16 mutants compared to the parental strain or
the genomically restored isolate (AUC = 628 vs.
155,237 or 148, 810) and the peak viral titers detected on day 4 in VP5/VP16
infected TG was more than 2 orders of magnitude lower than those detected in
17syn+ or VP5/VP16-1R infected TG (Figure 1D). The viral feedback loop between
the surface and the ganglion is well documented [38],[39].
The decline in viral titers in the eyes of VP5p/VP16 infected mice (days
6–8) most likely resulted from the absence of significant replication
in the ganglia and transport of virus back to the eye as described previously
[39]. Importantly, the high viral titers generated by
mutant VP5p/VP16 in RSC and on the corneal surface in vivo confirm the
infectious nature of the virions, which would not be the case if levels of VP16
in the tegument were deficient [30],[40],[41].
The VP16 protein produced during infection with this mutant is fully functional
and would be anticipated, if it were indeed efficiently transported to the
neuronal cell body, to initiate lytic viral infection in the neuron. However,
the replication of the VP5/VP16 mutants in TG is severely impaired, although
viral DNA is transported to the ganglion as determined by real-time PCR assay
(not shown). This strongly suggests that viral replication in neurons is in fact
not initiated by VP16 protein transported from the surface,
but rather by its synthesis in the infected neuron de novo. The profound
selective loss of replicative capacity in the TG of mice infected with the
VP5p/VP16 mutant provides the first evidence that the VP16 promoter is unique in
its ability to regulate gene expression in the nervous system and supports the
hypothesis that VP16, through distinct regulation in the TG neurons, could play
an important role in exiting latency.

(A) A diagram of the construction of the VP5p/VP16 mutants is shown. The
VP16 promoter and 5′UTR sequences were replaced with those of
another gene expressed with leaky late kinetics, the VP5 gene, as
detailed in Methods. (B) RSC were
infected with mutants VP5/VP16-1 and -3, the genomically restored
isolate (VP5p/VP16-1R), and wild type HSV-1 strain 17Syn+ at an
moi of 0.0004 pfu/cell. At the indicated times, 3 plates infected with
each virus were harvested and assayed independently for virus content as
detailed in Methods. (C,D) Mice
were infected as detailed in Methods and, at the indicated times pi, tissues from three mice
from each group were assayed for virus content. The grey shading in C
and D indicates the regions employed to calculate the areas under the
curves for the VP5p/VP16 mutants.

Altered expression kinetics of the VP16 promoter in neurons in vivo

The possibility that de novo expression of VP16 may be required in neurons during
both the acute stage of infection and during reactivation is suggested when
considering collectively (i) the well documented requirement for VP16
transactivating function during the acute infection in TG [16],[30]
(presumably for entry into the lytic cycle) , (ii) the inadequacy of leaky late
expression of VP16 from the VP5 promoter to support lytic viral replication
(reported here) and (iii) evidence from another α-herpes virus that
viral nucleocapsids arrive at the neuronal cell body largely devoid of VP16
[42],[43]. Framed within
conventional understanding of HSV gene regulation, the question to be asked is
straightforward, namely is VP16 expressed as a late gene, as demonstrated in
tissue culture or is VP16 expressed with distinct kinetics in neurons? The
concept of cascade gene regulation [44] and the kinetic
class of viral promoters during viral lytic cycle are fundamental to how we view
this process. However, these criteria were developed from en masse analyses of
synchronously infected cells of uniform type in the presence of drug blockades.
This experimental format cannot be recapitulated in vivo. One approach to
evaluating promoter activity in vivo is through the generation of viral
promoter/reporter mutants [45]–[55]. For this
purpose, a VP16 promoter/beta-galactosidase gene (LacZ) reporter mutant was
generated as detailed in methods and utilized to ask whether activation of the
VP16 promoter in neurons is consistent with conventional leaky late gene
expression. If this were the case, then VP16 promoter activity would be
anticipated only in neurons expressing lytic viral protein.

TG from mice inoculated with 2×105 PFU of 17VP16pLZ were
harvested on days 4 and 5 pi and processed sequentially for in situ E. coli
beta-galactosidase (b-gal) activity and for HSV proteins as detailed previously
[25],[28],[55].
Figure 2A shows two
populations of neurons evidencing activity from the viral genome. In the
majority of these neurons (464/551, 86%), VP16 promoter activity was
co-localized with lytic viral proteins. However, in 13–16%
of positive (infected) neurons, the VP16 promoter was active in the absence of
detectable lytic viral proteins. Even if very low and undetectable levels of
viral proteins are present in these neurons, the findings indicate that in
neurons, activation of the VP16 promoter can precede expression of significant
levels of viral proteins, an expression pattern inconsistent with our
understanding of late gene expression.

Figure 2. Activation of the VP16 promoter in sensory neurons during acute
infection.

(A) Mice were infected with 17VP16pLZ. At the indicated days pi, TG were
removed and sequentially processed for the detection of b-gal activity
(blue) and viral proteins (purple), as detailed in Methods. A photomicrograph of a focus of infection
in a TG is shown. White arrows indicate neurons with b-gal activity with
little or no detectable viral protein. Counts of neurons positive for
only b-gal or b-gal plus viral protein are indicated below the
micrograph. (B) Photomicrographs of viral plaques on RSC monolayers
infected at low moi with 17-0pZ563gJ and 17VP16pLZ are shown. The
monolayers were stained to detect b-gal and viral proteins. At regions
at the edge of the plaques formed by 17-0pZ563gJ cells expressing b-gal
(blue) with little or no evidence of viral protein expression were
evident (arrow). Regions at the edge of plaques formed by 17VP16pLZ show
cells staining positive for viral proteins (purple) with little or no
staining for b-gal evident (arrow).

When examined in infected RSC, the pattern of expression of the VP16 promoter was
consistent with late kinetics in that at either high or low multiplicity of
infection (moi), b-gal activity was detected only in cells in which viral
proteins were also detected. The asynchrony of low moi infection more closely
represents infection in vivo and in this case plaques formed by 17VP16pLZ were
ringed by cells expressing viral proteins but little or no b-gal activity (Figure 2B). We examined the
expression of an IE gene promoter/reporter virus, 17-0pZ56gJ [55]
using this same assay and observed that plaques were now ringed by cells
expressing b-gal with very low levels of viral proteins present, as would be
expected for a promoter activated at the initiation of the lytic cycle. These
findings support the hypothesis that the regulation of VP16 in vivo is dependent
on cell type and different from that seen in vitro.

VP16 is expressed during reactivation in the absence of ICP0, ICP4, and viral
DNA synthesis

We have reported previously that two viral functions (ICP0 and viral DNA
synthesis) considered to play critical roles in the initiation of reactivation
from latency, are in fact not required for lytic viral protein expression
following a reactivation stimulus in vivo [25],[28].
These functions are, however, required for progression to infectious virus
production. This knowledge provides the opportunity to ask whether VP16 is
expressed in the absence of ICP0 function and in the absence of viral DNA
replication following a reactivation stimulus. If VP16 is not present, it would
suggest that this protein is not likely to be initiating entry into the lytic
cycle. If, however, VP16 is detected, it would be consistent with an early role
and reveal that in the context of reactivation, the expression of VP16 is not
dependent upon either ICP0 function and/or viral DNA replication, both of which
play a role in the regulation of late gene expression in cultured cells.

The exit from latency in vivo is highly a controlled process, restricted to a
very small percentage of those neurons latently infected per event. Despite
this, the number of neurons exiting latency and the number of neurons expressing
VP16 can be quantified using whole ganglion immunohistochemistry (WGIHC), an
assay that has been validated to provide a precise quantitative readout on the
number of neurons expressing lytic viral proteins within a ganglion [56]
Groups of mice were inoculated with either dl1403 (an ICP0 null mutant [57]) or
17tBTK- (a thymidine kinase negative mutant [24]). In the absence
of the viral thymidine kinase (TK) function, viral DNA synthesis and replication
in neurons are severely impaired. This gene is required for reactivation [58]–[61] but not for entry
into the lytic cycle from the latent viral genome [28]. The deficit in
each of these mutants results in significantly reduced total latent viral DNA
[62]–[64] and numbers of
latent infections in the TG, [25],[28],[65],[66], which in turn,
reduces the number of neurons which exit latency [25],[28],[55],[56],[66]. Nevertheless,
VP16 protein was detected at 22 hrs post hyperthermic stress (HS) in neurons in
ganglia from mice latently infected with both of these mutants (3/27 and 9/20 in
17tBTK- and dl1403 infected ganglia, respectively). Analysis of the second TG of
each pair with the anti-HSV antibody revealed no difference compared to the
number of neurons in which VP16 was detected (4/27 and 8/20 in 17tBTK- and
dl1403 infected ganglia), and numbers similar to our previous reports [25],[28]. Viral protein
expressing neurons were not detected in uninduced latently infected ganglia
(0/18). The number of neurons expressing VP16 within a positive ganglion ranged
from 1–3, and these numbers were not different than those detected
when using the anti-HSV antibody, which detects lytic viral proteins from IE,
early (E) and late (L) kinetic classes. Likewise, following HS of mice latently
infected with tsK+, a 17syn+ based mutant with a temperature
sensitive mutation in the essential viral IE transactivator ICP4 [67], VP16
was expressed in rare neurons. The number of neurons in which latency was
established with this mutant was very low (2.7%), yet VP16 was
detected in rare neurons post stress (2 neurons in 2/30 ganglia were positive).
This number is consistent with the frequency of reactivation observed in ganglia
infected with wild type 17syn+ in which there were similar low levels
of latency (1/31 positive) [56]. These findings indicate that independently,
neither ICP0, ICP4, nor viral DNA replication is required for VP16 expression
during reactivation in vivo.

In order to examine a larger population of neurons exiting latency, a chemical
blockade of viral DNA replication was used to investigate the influence of viral
DNA replication on the expression of VP16 in 17syn+ infected neurons
following a reactivation stimulus. As shown previously, acyclovir (ACV) blocked
detectable infectious virus production during reactivation in vivo [28],[68]. Infectious virus
was not detected at 22 h post HS in the 14 TG tested (0/14). Ganglion pairs from
an additional 15 mice from this group were harvested and examined using WGIHC.
The number of neurons expressing lytic viral proteins of diverse kinetic classes
was quantified in one ganglion from each pair and VP16 expression was quantified
in the second ganglion from each pair. The number of neurons exiting latency,
whether detected by the anti-HSV antibody (31 neurons/15 ganglion) or the
antibody specific for VP16 (33 neurons/15 ganglion) was not different and
similar to the numbers of neurons exiting latency previously reported [4],[24],[25],[55],[56],[66],[69].
As observed in 17tBTK- infected TG, blockade of viral DNA replication did not
alter the expression of VP16.

Mutants lacking the transactivation function of VP16: in1814 and
17VP16Δ422

VP16 is an essential multifunctional protein. However, mutations which impair the
transactivation function of VP16 have been generated and characterized in vitro
and in vivo. Mutant in1814 contains a 12 bp insertion that disrupts a domain
required for the VP16 induced complex formation and thus the transactivation
function of the protein [40],[70]. The carboxy-terminal acidic activation
domain has been deleted in two mutants, V422 [41] and RP5 [30],
both built in HSV-1 strain KOS. While these three mutants are phenotypically
similar in vitro, important differences have been reported in their in vivo
phenotypes. Despite the impaired replication reported for both in1814 and RP5 in
mouse eyes and TG, in1814 established latent infections efficiently and
reactivated in explant assays [16],[71],[72]. RP5 failed to accomplish either of these
outcomes [30]. The in vivo phenotypic differences between
HSV strains 17syn+ and KOS is a confounding issue [69],[73].
Therefore, mutant 17VP16Δ422 was constructed as detailed in methods. We utilized mutants in1814 and
17VP16Δ422 to evaluate the role of VP16 transactivation on reactivation
in vivo as outlined in Figure
3A.

(A) A schematic of the experimental design employed to biologically
characterize the VP16 mutants is shown. (B) A diagram of the genomic
structures of in1814 and 17VP16Δ422 is shown. (C,D) Mice were
infected as detailed in Methods
and, at the indicated times pi, tissues from three mice from each group
were assayed for infectious virus. Solid lines represent titers obtained
by standard plaque assay, whereas dashed lines represent the same
samples titrated in the presence of 3 mM HMBA, as described in Methods. The area under the curve
(AUC) was calculated for each curve, and the fold increase in AUC in the
presence of HMBA is indicated below the graphs. Genomically restored
isolates of both in1814 (1814R) and 17VP16Δ422
(17VP16Δ422R) were analyzed and found to be not different from
the parental strain, 17syn+ (not shown).

In vivo replication kinetics

Groups of male Swiss Webster mice were inoculated as described in methods. Viral replication was evaluated on
days 2,4,6,8 and 10 pi in tissues harvested from 3 mice from each inoculation
group. On day 4 pi, 3–4 logs fewer pfu were detected in the eyes and
TG of in1814 and 17VP16Δ422 infected mice compared to those infected
with in8141R, 17VP16Δ422R, and 17syn+ (Figure 3C and 3D, and not shown). These
results are in general agreement with previous reports [16],[30].

Interpretation of this result is complicated by the fact that at low moi (such as
a plaque assay), mutants lacking the transactivation function of VP16 enter the
lytic cycle inefficiently, leading to an underestimate the amount of virus
present [40],[41],[74].
Several strategies have been utilized to overcome this problem, including VP16
expressing cell lines, and superinfection with a replication impaired virus
[16],[30]. The addition of
the cell differentiating agent, hexamethylene bisacetamide (HMBA), to cell
cultures increases the plaquing efficiency of in1814 [74]. As shown in Figure 3C and 3D, the addition
of HMBA to the culture medium revealed the presence of 100 and 500 fold more
virus in in1814 and 17VP16Δ422 eye homogenates (day 2 pi), respectively.
As anticipated, this compound had little effect on the plaquing efficiency of
the parental strain, 17syn+ (1.8 fold increase in virus detected in
homogenates from 17syn+ infected eyes). Differences between the two
VP16 mutants, in1814 and 17VP16Δ422, to replicate in the TG were
dramatic. Plaque assays performed in the presence of HMBA revealed that in1814
did replicate within the TG, although maximum titers are 17 fold lower than
those achieved by wild type virus or 1814R (Figure 3D and not shown). In contrast, even
in the presence of HMBA, infectious virus was not detected in 17VP16Δ422
infected TG, although ∼200 pfu were detected in the TG on day 4 pi when
input titers of this mutant were increased (not shown). The importance of viral
replication within the TG for achieving maximum numbers of latently infected
neurons has been demonstrated [66]. That in1814 actually does replicate within
TG is consistent with its ability to efficiently establish latent infections as
well as reports that this mutant may retain some residual transactivating
function [75].

Quantification of the number of latently infected neurons and latent viral
genome copy number profile in in1814-infected ganglia

In preliminary studies, 17VP16Δ422 was determined to establish latent
infections, but at very low levels compared to 17syn+ (not shown). Thus
it becomes impractical to study in vivo reactivation with this mutant because
the efficiency of reactivation in vivo following HS is directly correlated with
the number of latently infected neurons in the ganglion
(r2 = 0.99) [56],[76].
Likewise quantification of the number of latently infected neurons in in1814
infected TG compared to the parental strain (17syn+) and rescue (1814R)
is critical for interpreting the outcome of experiments to quantify viral
reactivation. The number of latently infected neurons and the number of viral
genomes within individual infected neurons in TG from 3 mice from each group was
quantified using a single neuron PCR assay termed CXA [25],[28],[66],[77]. In this assay,
ganglia stabilized by fixation are enzymatically dissociated and individual
neurons from enriched neuronal fractions are harvested and analyzed by QPCR,
providing information on both the frequency of latently infected neurons and the
number of viral genome copies in the individual neurons analyzed. As anticipated
from the results of preliminary experiments, similar numbers of latently
infected neurons were observed in in1814, 1814R, and 17syn+ infected
ganglia, 28%, 25% and 26%, respectively (Figure 4A). The number of
viral genomes detected within individual latently infected neurons is shown in
the scattergram (Figure 4B).
No significant difference among the viral genome copy number profiles was
observed (mean copy number = 56.5, 51.8 and
44.8, respectively p = 0.94; ANOVA).

Groups of mice were infected with strain 17Syn+, in1814, and the
genomically rescued variant 1814R, as described in Methods. At 40 days pi, the ganglia of 3 mice per
group were processed for single neuron PCR. Individual neurons were
examined for the presence of the viral genome and the number of viral
genomes present in positive neurons was determined using a quantitative
PCR assay as detailed in Methods.
(A) Shown is the percentage of neurons positive for the viral genome.
The number of neurons positive for the viral genome over the number
tested is shown in the histograms. (B) Each point on the scattergram
represents the number of viral genomes present in an individual neuron.
The horizontal bars are drawn at the mean value of genome copies per
positive neuron.

In1814 does not reactivate in vivo following HS

At 40 days pi, mice from each group of infected mice were induced to reactivate
in vivo using HS [5],[24]. At 22 hrs post
treatment, mice were euthanized, ganglia were removed and homogenized and the
homogenates assayed for infectious virus in the presence of 3 mM HMBA. In
striking contrast to previous reports in which in1814 reactivated in explant
reactivation assays, infectious virus could not be detected in any ganglia
(0/20) from in 1814 infected mice induced to reactivate in vivo. However,
infectious virus was detected at 22 hr post treatment in 17/20 (85%;
p = 0.0002) and 16/20 (80%;
p = 0.0002) of the TG pairs from mice infected
with 1814R and 17Syn+ (Figure 5A).

Figure 5. The mutant in1814 does not reactivate from latency in vivo but does
reactivate in explanted ganglia.

(A) Mice were infected as described in Methods. At 40 days pi, latently infected mice were induced
to reactivate using HS, and TG were removed and assayed for infectious
virus as detailed in Methods. The
histograms represent the percentage of mice in which reactivated HSV was
detected. The number of mice positive/tested is shown on the histograms.
(B) Explant reactivation of ganglia latently infected with in1814 or
17VP16Δ422. TG from latently infected mice were axotomized and
explanted. In1814 and 17VP16Δ422 infected TG were assayed for
infectious virus on days 5 and 15 post explant, respectively. The GFP
expression cassette in 17VP16Δ422 was used to detect gene
expression from the viral genome during explantation. Exit from latency
was observed as single GFP positive neuron at day 4 (C) which resulted
in the spread of virus through the TG by day 6 (D).

HS has been utilized extensively and shown to reproducibly induce viral
reactivation which peaks at ∼22 hrs post treatment using several
laboratory strains [69] as well as 10 low passage clinical isolates
(unpublished). However, it was possible that reactivation of in1814 was delayed
compared to wild type virus. Infectious virus could not be detected in ganglia
of in1814 infected mice 48 hrs post HS (0/20). These data demonstrate that
in1814 did not reactivate to detectable levels in vivo in response to HS.

In1814 and 17VP16Δ422 reactivate in vitro following explant of TG
into culture

In order to confirm that ganglia from this group of in1814 infected mice would
produce infectious virus when axotomized and explanted as previously reported
[16],[17], the 10 TG from
5 mice latently infected with in1814 were either directly homogenized and
assayed for infectious virus in the presence of HMBA or explanted and cultured
for 5 days and then tested for infectious virus in the presence of HMBA (see
methods). No virus was detected in TG
homogenized directly upon removal but infectious virus was detected in
100% (5/5) of explanted TG, a finding similar to previous reports in
which neurons were axotomized [16],[17] (Figure 5B). Similarly, the
ability of ganglia from 17VP16Δ422 infected mice to reactivate in
explant was tested. The presence of eGFP in this virus was used to monitor exit
from latency and spread within the explanted ganglia over time. No GFP
expression was detected in ganglia (0/6) at the time of explant, however within
4 days, a single GFP positive neuron was detected in 1/8 ganglia and by day 6
post explant, virus had spread within this TG (Figure 5C and 5D). After 15 days in explant,
17VP16Δ422 exited latency in 4/6 ganglia (Figure 5B). Ganglia were homogenized and
plated in the presence of HMBA and infectious virus was recovered which was GFP
positive and confirmed by southern blot to have the expected genomic structure
(not shown).

Reactivation from latency is functionally defined by the detection of infectious
virus. To expand our understanding of the process of reactivation, we have
developed a strategy for quantifying at the single neuron level the number of
neurons which exit latency as evidenced by detectable lytic viral protein
expression [4],[78]. This method is
the first to allow us to partition the process of reactivation into stages, and
to begin the assignment of viral and host cell functions critical for either
entry into the lytic cycle or for progression to infectious virus production
[25],[28]. In addition, this
approach obviates the inherent problem of detection of reactivated virus when a
mutant with low plaquing efficiency (such as in1814) is employed. At 40 days pi,
additional mice from the groups detailed above were induced to reactivate in
vivo using HS. Latently infected control mice and treated mice (at 22 hrs post
treatment) were euthanized, the ganglia were removed and processed for the
detection of lytic viral proteins using WGIHC as detailed previously [4],[78]. This method can
reliably detect a single neuron exiting latency among the 10's of
thousands in the ganglion. In the uninduced groups of animals, lytic viral
protein expressing neurons were not observed (0/10, 0/8, and 0/9, in1814, 1814R
and 17syn+ infected ganglia, respectively). This was not unexpected as
we have previously shown that the level of “spontaneous”
reactivation of strain 17syn+ in the latently infected Swiss Webster
mouse TG is very low [4],[78]. Consistent with
the detection of infectious virus above, ganglia from mice latently infected
with either 1814R or 17syn+ contained neurons expressing lytic viral
proteins at 22 hrs post induction, a total of 60 and 55 neurons, respectively in
the ganglia examined (Figure 6A and
6B). In contrast, no lytic viral protein expressing neurons were
detected in in1814 ganglia post induction (0/40). These findings indicate that
in vivo, the VP16 transactivating function is required for HSV to exit the
latent state and produce detectable viral proteins.

(A) Mice latently infected with the indicated strains were subjected to
HS, and whole TG were assayed for viral protein positive cells 22 hrs
post stress. Each point in the scattergram represents the number of
viral protein positive neurons detected in an individual TG. The
horizontal bars are drawn at the mean. (B) A photomicrograph of a whole
mounted ganglion processed for the detection of HSV-1 proteins 22 hr
post HS shows a single neuron in which the latent genome has entered the
lytic cycle. The brown precipitate evident in the cell body and axonal
tract indicates the presence of viral proteins.

If VP16 functions at the earliest stages of the initiation of reactivation, its
role is likely to be the coordinated activation of immediate early genes through
the TAATGARAT promoter elements. We reported previously that mutant
ΔTfi, which contains a 350 base pair deletion in the ICP0 promoter
including the TAATGARAT element, reactivates with wild type frequency and
kinetics from ganglia axotomized and explanted [25],[29],
but exhibits severely impaired reactivation in vivo [25]. To test the
importance of the TAATGARAT to this phenotype, a mutant,
ΔTfi+TAATGARAT, in which the TAATGARAT regulatory element was
added back in its proper context to the ICP0 promoter of ΔTfi, was
generated as detailed in methods and tested for its ability to reactivate in
vivo.

Mice were inoculated with 4×106 pfu of ΔTfiR or
ΔTfi+TAATGARAT and at 40 days pi groups of latently infected
mice were utilized to quantify the number of latent infections and to determine
the in vivo reactivation frequency. There was no difference in either the number
of neurons latently infected or the viral genome copy number profile in TG
latently infected with ΔTfiR or ΔTfi+TAATGARAT (not
shown). Infectious virus was not detected in latently infected uninduced TG from
either ΔTfiR or ΔTfi+TAATGARAT infected mice, 0/5 and
0/5, respectively. However at 22 h post HS, infectious virus was detected in
41% (7/17) of TG from ΔTfiR and 44% (8/18) of TG
from ΔTfi+TAATGARAT infected mice. Taken together, this finding
and the clear requirement for VP16 transactivating function for the exit from
latency in vivo, support the hypothesis that as during acute lytic infection,
VP16 operates through the TAATGARAT element to regulate immediate early gene
expression during reactivation.

VP16 promoter is activated in neurons latently infected with in1814 following
HS

The preceding experiments suggest an amplification feedback loop between VP16 and
the IE gene products is required to exit latency. If this is true, expression of
VP16 must occur very early, requiring that the VP16 promoter be activated de
novo in contrast to the standard cascade of viral functions facilitating the
activation of the viral leaky late promoters. With wild type HSV, once
reactivation is initiated, the production of the viral IE transactivator
proteins induces transcription from all viral promoters in a cascade fashion.
That in1814 fails to produce detectable viral proteins following HS of latently
infected ganglia affords a unique opportunity to directly examine VP16 promoter
function in the absence of other viral proteins. Thus activation of the VP16
promoter following a reactivation stimulus in neurons latently infected with
in1814 would provide strong support that transcription of the VP16 gene can be
upregulated in the absence of other viral proteins during the earliest stages of
exit from latency. To test this hypothesis mutant in1814VP16pLZ was generated as
detailed in methods. The in vivo phenotypes of this mutant (replication,
establishment of latency and reactivation) were not different than in1814 (not
shown). Mice were infected with in1814VP16pLZ and maintained for 20 days pi.
Groups of mice were subjected to HS and at 22 hr post HS ganglia were removed
and processed to detect b-gal activity. No neurons were positive in 20 TG from
infected untreated animals. However, 5/20 of the ganglia from treated mice
contained one or more neurons positive for b-gal post HS, demonstrating that the
VP16 promoter had been activated in these neurons. In additional studies it was
observed that activation of this promoter post HS becomes increasingly rare as
time progresses which parallels the ability to detect VP16 protein (not
shown).

Discussion

Successful completion of the complex in vivo life cycle of both herpes simplex virus
type 1 and type 2 requires activation of the viral lytic cycle from the latent viral
genome. Since HSV latency is characterized by the absence of all detectable lytic
viral proteins, the lytic cycle must start de novo, i.e. without the viral proteins
normally carried into a cell. These proteins, among other functions, facilitate
activation of transcription from the viral genome. Studies performed nearly two
decades ago led to the formative conclusion that VP16 does not
coordinate the exit from latency [15],[16],[72]. Using new
approaches we have revisited this important issue and now demonstrate that the
transactivating function of VP16 is indeed requisite for HSV reactivation in vivo.
The functional requirement for VP16 is very early in the transition from the latent
to the lytic cycle, as in its absence the latent viral genome cannot advance to the
production of lytic viral proteins. In contrast, elimination of “immediate
early” and “early” viral functions previously
considered critical for the initiation of reactivation has no measurable effect on
lytic viral protein expression at this early stage in reactivation 25,28. We show that replacing the VP16 promoter
with another leaky late gene promoter exposes unique regulatory properties of the
VP16 promoter in the context of infection in neurons in vivo. The VP16 promoter is
responsive to reactivation stimuli in the absence of other viral functions and
robust expression of VP16 from the latent viral genome is observed in neurons in the
absence of viral functions normally required for efficient late gene expression.
These data argue that the expression of VP16 during reactivation diverges from the
established late expression pattern of this gene. Our findings lead to the
hypothesis that in the context of the latently infected neuron, the expression of
VP16 is a critical initiating event, coordinating the activation of the viral IE
genes which results in productive entry into the viral lytic cycle.

The differing outcomes and thus conclusions regarding the role of VP16 in
reactivation stem primarily from the models of reactivation utilized. In previous
studies utilizing in1814 in the mouse ocular or footpad models reactivation was
evaluated ex vivo by removing ganglia from the latently infected animal and
culturing the tissue (explanting) for >21 days [16],[17]. Recent studies
demonstrate that axotomy and explant result in rapid and progressive changes in the
gene expression and physiological states of neurons and other cells not observed in
ganglia following an in vivo stress resulting in reactivation [24]. These changes can
obviate the need for and obscure the roles of viral genes important for the
reactivation process [25]. O'Hare suggested that VP16 might indeed
play a pivotal role during the early stages of reactivation, proposing that
explantation of ganglia into culture might overcome the requirement for VP16 [26], thus
explaining reports that in1814 reactivated ex vivo [15]–[17].
Our results confirm this hypothesis.

Quantitative analyses of both latency and reactivation at the single neuron level
have increased understanding of the relationships between viral replication, the
number of latently infected neurons, and the probability of reactivation [4],[25],[27],[28],[56],[69],[77],[79].Viral
replication within the trigeminal ganglia (TG) is required for maximizing the number
of latent infections [66] and mutants that replicate poorly in the TG
generally establish latency very inefficiently [25],[28]. In previous studies
mutant in1814 seemed to be an exception as no replication of in1814 was detected in
sensory ganglia [16],[72] and yet latency was
established very efficiently as determined by the detection of the latency
associated transcripts [16],[72]. However, with the
addition of HMBA to the plaque assay, it is clear that in1814 does replicate in the
TG. The mutant 17VP16Δ422, however, is severely restricted in TG and
establishes very low levels of latency as would be predicted by its limited
replication. It seems likely that mutated VP16 protein produced by in1814 still
retains some ability to transactivate IE genes, perhaps through elements other than
the TAATGARAT. VP16 has been shown to transactivate through the GCGGAA element in IE
gene promoters and this activity is independent of transactivation through the
TAATGARAT element [80],[81]. Despite the low
levels of latency established by 17VP16Δ422 this mutant did reactivate in
67% of the ganglia placed into explant culture within 15 days, a finding
that further emphasizes the differences inherent in the in vivo and ex vivo
reactivation models.

In vivo reactivation is a tightly regulated event in that following induction the
lytic cycle is engaged in ∼0.05% of neurons harboring the latent
genome [4]. Viral infection is confined to the individual neurons
undergoing reactivation (usually 1–5 neurons per TG) with no spread of
infection within the ganglion and consistent with this, it is characterized by low
levels of infectious virus in the ganglion [3],[5],[82]. Reactivation of HSV in
vivo has been characterized most extensively following hyperthermic stress, however
other induction triggers, including ultraviolet light irradiation [3],[6] show a
similar outcome. In contrast, in explanted ganglia virus infection spreads
unchecked, accounting for the extremely high titers of virus recovered [24]. The
limited production of virus in vivo raises the issue of sensitivity and the
possibility that the differing reactivation outcomes observed merely reflect
differences in detection of the infectious virus produced in the ganglia, a
consequence of reduced plaquing efficiency of in1814 compared to 1814R or
17syn+ [40]. This question could not be resolved without a
different approach for detecting the exit from latency, one that did not depend on
the detection of infectious virus. The need for such an assay has long been
recognized [31],[83],[84] and an in situ immunohistochemical method in
whole ganglia that permits the detection of lytic phase viral proteins in the very
rare neurons that exit latency was employed for this purpose [4],[24],[25],[28],[55],[78]. This assay makes it
possible to evaluate the ability of viruses that are blocked or impaired at later
steps in the replicative cycle, to exit latency. This assay revealed that the
failure to detect infectious virus in the ganglia latently infected with in1814
following induction was not merely an issue of sensitivity but an actual failure of
latent in1814 genomes to exit latency. The implications of this observation are
profound in that our attention is directed toward the regulation of VP16 from the
latent genome as a critical interface between neuronal responses to stress and entry
into the lytic cycle.

An important question relating to the mechanism of reactivation is whether this
phenotype is unique to VP16 transactivating function or whether other viral
functions are required at this earliest stage in the reactivation process. Two
hypotheses framed thinking about how the lytic cycle was initiated from the latent
genome without VP16 function. The first suggested that ICP0 served this function
[18]–[21] and the second proposed
that limited viral DNA replication is required for and precedes efficient expression
of IE genes [22],[23]. Using the same approaches utilized for the
analysis of in1814, the role of these viral functions in reactivation were examined.
In the absence of ICP0 function, initiation of reactivation as evidenced by lytic
viral protein expression was not measurably different from that in the rescue or
parental strain. Thus in contrast to VP16 transactivating function, ICP0 is not
essential for these very early events. A similar result was observed when viral DNA
replication were blocked either pharmacologically or genetically. In both cases,
viral proteins but not infectious virus was detected, confirming the roles of these
viral functions for the progression to infectious virus production [25],[28]. These
studies emphasize that VP16 appears to play a unique role in the earliest stages of
reactivation to coordinate IE gene expression and thereby entry into the lytic
cycle. Further evidence that VP16 functions early in the process of reactivation to
upregulate IE genes through the TAATGARAT motif comes from our finding that adding
back the TAATGARAT sequences to the ICP0 promoter in mutant ΔTfi restored
the ability of this mutant to reactivate in vivo. This is in keeping with a
requirement for ICP0 for progression to infectious virus production and the
TAATGARAT element for proper expression of ICP0 during reactivation in vivo.

The extreme rarity of viral reactivation at the neuronal level might be explained if
stress induced changes occur only in very rare neurons, or if only a few latently
infected neurons contain viral genomes capable of reactivation, but neither is the
case. Stress does induce global changes in the chromatin structure of latent viral
genomes [85]–[90]. To be detected such
changes must occur on the majority of latent viral genomes, while only a very few
viral genomes exit the latent state. Thus, the alterations in chromatin measured may
or may not be necessary but are not sufficient to precipitate viral
reactivation. Many latently infected neurons are capable of reactivation as
evidenced by semi-quantitative assays performed on dissociated latently infected
ganglia [91]. Likewise, over a period of a few days, 100s of
neurons produce viral proteins (exit the latent state) in TG axotomized and
explanted into culture in the presence of the antiviral drug acyclovir (which
prevents virus replication and spread within the TG) [24],[28]. Further, we have
determined that viral reactivation can be induced repeatedly (30 times over 10
months) in the same mice in vivo without reduction in the frequency of reactivation
(unpublished).

Reactivation from latency has been traditionally thought to be the result of changes
in host neuronal transcriptional regulators induced by systemic stress which then
directly stimulate the activation of one or more viral promoters. However, it is
difficult to reconcile the extreme rarity of these reactivation events with this
simple genetic model. For this model to be correct the stress induced signals that
initiate reactivation from latency must only occur in very rare neurons at any given
time, and since neurons do not survive viral reactivation [3],[5],[78], these same rare
changes would have to occur in a new rare subset of neurons as each virus
reactivation event occurs through time. The extremely low frequency of reactivation
suggests a model of stochastic derepression of VP16 in the presence of positive
transcription factors [92]. This type of model would predict that the
transcription and/or translation of viral genes is extremely repressed and that
viral reactivation is essentially an extremely rare and seemingly random event
precipitated when the amount of VP16 protein present in a given neuron reaches a
level adequate to initiate the cascade of lytic viral gene expression.

The hypothesis that VP16 functions in conjunction with host cell proteins as a
regulatory switch, promoting the lytic cycle when VP16 is present and latency in its
absence has been proposed by many investigators [13],[15],[26],[32],[83],[93]. Sears and Roizman first
proposed that the VP16 protein in the tegument may be inefficiently transported the
distance through the axon to the cell body, thereby promoting latency [15].
Although their early attempts to support this hypothesis experimentally were not
successful, there is support for the notion that the VP16 equivalent in pseudorabies
virus is dissociated from the nucleocapsid prior to transport to the neuronal cell
body [43].
Importantly, our results imply that VP16 generated and packaged into virions during
surface replication is not sufficient to efficiently activate IE genes in TG
neurons.

We propose that neuron driven regulation of VP16 orchestrates its de novo synthesis
which is central to coordinated entry into the lytic cycle in neurons and the
balance between the lytic and latent viral programs (Figure 7). During acute infection, virus
replication at the body surface feeds virions into the axons of innervating sensory
neurons (Figure 7A). Data
support the notion that VP16 is not transported efficiently to the neuron nucleus
and in its absence the latent transcriptional pathway is engaged. De novo expression
of VP16 is then required for the virus to enter the lytic pathway in these neurons
and VP16 is only expressed when repressors are overcome. Repressors that operate
during the acute stage of infection are likely to include riboregulators encoded by
the latency associated transcript locus (LAT) [94]–[96].
Expression of the LAT locus is required for ∼65% of the latent
infections established [27],[79], and in the absence
of LAT transcription, half of the neurons destined to be latently infected enter the
lytic pathway and die [27]. If sufficient VP16 is expressed to coordinate IE
gene expression, a positive feedback loop overcomes repression, the neurons becomes
lytically infected and the virus produced spreads both within the ganglion as well
as back down to the surface. Efficient virus replication at the surface and within
TG is required to maximize the number of latent infections established [66].
Acute virus replication ends by about 10 days pi and during the period between 10
and 40 days pi latency becomes consolidated (Figure 7B), perhaps through chromatinization of
the viral genome [85], [88]–[90],[97],[98], the recently proposed
immune mediated non-lethal inhibition of virus production once the exit from latency
has been initiated in neurons [99], and/or the build up of riboregulators [94]–[96],[100],[101], and fewer latently
infected neurons respond to stresses that induce viral reactivation [4]. By 40
day pi about 0.05% of latently infected neurons show evidence of viral
reactivation following HS whereas latency is maintained in the other
99.95% [4],[56],[69]. We hypothesize that stress induces the de novo
production of VP16, which only rarely reaches levels sufficient to coordinate
activation of the IE genes that overcome repressive factors and initiate the lytic
transcription program.

Figure 7. The regulation of expression of VP16 controls the balance between lytic
and latent infection of sensory neurons at all stages of the virus infection
cycle.

(A) Shown is a schematic of the role of de novo VP16 expression during acute
infection of the trigeminal ganglion. As detailed in the text, we
hypothesize that VP16 protein is not transported efficiently to neuronal
cell bodies and that sequences present in the VP16 promoter direct the de
novo expression of VP16, which overcomes repressors (e.g. riboregulators) to
initiate lytic infection during the acute stage of viral replication. (B) We
hypothesize that following stress the de novo synthesis of VP16 initiates a
feedback loop with the IE genes that results in viral reactivation in one or
a very few of the 6,000 latently infected neurons present in the
ganglion.

Materials and Methods

Viral strains/mutants and stock production

Stocks of HSV-1 strain 17syn+ and the mutants employed in this study
were generated in rabbit skin cell (RSC) monolayers and the viral titers were
determined by serial-dilution plaque assay [5],[102].
The wild type HSV-1 strain 17syn+ was originally obtained from John H.
Subak-Sharpe at the MRC Virology Unit in Glasgow, Scotland. The generation and
characterization of the VP16 transactivation deficient mutant in1814 and its
genomically restored counterpart, 1814R, have been described [16],[40]. Where indicated, the plaquing efficiency of
in1814 was enhanced by the addition of 3 mM hexamethylene bisacetamide (HMBA,
Sigma) as described [74],[103]. The mutant
ΔTfi and its genomically restored counterpart, ΔTfiR were a kind
gift of David Leib, Washington University, and have been described in detail
[29],[104].

Construction of new viral mutants

All restriction enzyme sites and base pair numbering are referred to as the
corresponding positions in the published HSV-1 sequence of strain
17syn+ [105],[106] as present in
Genbank (NID g1944536).

Mutant VP5p/VP16

A 649 bp DNA molecule was synthesized (Blue Heron) in which the VP16 promoter and
5′UTR (bp 105,435 to 105,107) was converted to the promoter and
5′UTR of VP5 (bp 40,659 to 40,812), flanked by appropriate sequences
and recombined into strain 17Syn+ The promoter structures of 5
independently derived mutants were confirmed by Southern blot analysis and DNA
sequencing (not shown). Wild type UL49-UL48 sequences were recombined with
VP5p/VP16-1 to generate the genomically restored variant VP5p/VP16-1R as
described [27],[79].

Mutant 17VP16Δ422

Mutant 17VP16Δ422 was generated in strain 17syn+ to be similar
to mutant V422, in which the carboxy-terminal acidic transactivation domain of
VP16 was deleted in strain KOS [107]. The eGFP gene driven by the b-actin
promoter was inserted at the SacI site at bp 103,808 in the VP16 ORF in the
orientation opposite to VP16, truncating the protein at amino acid 422 and
facilitating selection of the mutants. 3 mM HMBA was added to the culture medium
to increase the plaquing efficiency of the VP16 mutants [103]. Following low
MOI infection of RSC, mutants displayed a reduced capacity to replicate and
plaque which was increased 40 fold in the presence of HMBA. Wild type UL49-UL48
sequences were recombined with 17VP16Δ422 to generate the genomically
restored variant 17VP16Δ422R as described [27],[79].

Mutant ΔTfi+TAATGARAT

Truncation of the ICP0 promoter at −145 just prior to the TAATGARAT
element resulted in an ICP0 promoter that drives expression efficiently in cells
and tissues in vivo with the exception of TG neurons, where this promoter fails
to function [55]. Using this information, a construct was
generated that restored sequences to −172 in the ΔTfi ICP0
promoter including 27 additional bases (CGTGCATGCTAATGATATTCTTTGGGGG)
that contain the functional TAATGARAT of ICPO (underlined). This construct was
recombined into both ICP0 promoters in the mutant ΔTfi to generate the
ICP0 promoter mutants termed ΔTfi+TAATGARAT. Independently
derived mutants were generated and characterized and all replicated as well as
wild type in RSC and in mice in vivo (not shown).

Mutants 17VP16pLZ and in1814VP16pLZ

Mutants 17VP16pLZ and in1814VP16pLZ express the E. Coli beta-galactosidase gene
(b-gal) from a 423 bp promoter/5′UTR fragment of the VP16 gene
(105,108-105,534 bp) inserted into the intergenic region between glycoprotein J
(gJ) and gD in strain 17Syn+, or the VP16 transactivation mutant in1814
respectively. A single base mutation (G>A) was introduced at 138,045 on
the viral genome to generate an EcoRV site in the intergenic region between gJ
and gD. The promoter/reporter cassette (terminated by bi-directional SV40
polyadenylation signals) was cloned in the orientation opposite that of the
viral gJ and recombined in to the genome [55]. Six
independently derived mutants replicated as well as wild type in RSC and in mice
in vivo (not shown). Wild type levels of gJ and gD mRNA of the expected sizes
were produced by the mutants (not shown).

Inoculation of mice

All procedures involving animals were approved by the Children's
Hospital or University of Cincinnati Institutional Animal Care and Use Committee
and were in compliance with the Guide for the Care and Use of Laboratory
Animals. Animals were housed in American Association for Laboratory
Animal Care-approved quarters. Male, outbred, Swiss Webster mice
(22–25 grams in weight, Harlan Laboratories) were used throughout
these studies. Prior to inoculation, mice were anesthetized by intraperitoneal
injection of sodium pentobarbital (50 mg/kg of body weight). A 10 ul drop
containing the amount of virus as detailed in the text was placed onto each
scarified corneal surface. In some experiments the inoculum titer was altered to
achieve uniform levels of latent infections as previously detailed [56],[66]. In preliminary
experiments we determined that inoculation of mice with
2×105 pfu of strain 17syn+ or in1814R and
8×105 pfu of in1814 resulted in similar levels of
latency as shown in Figure
4B. In these preliminary studies mice infected with in1814 at
2×105 pfu were examined. In these mice as in those
receiving the higher imput titer no exit from latency was detected.

Replication in vivo

Mice infected as above were euthanized at the indicated times post infection and
tissues from three mice from each inoculation group were individually assayed
for virus as previously detailed [49].

Quantification of latent infections by contextual analysis of latency

Additional mice from the groups infected as above were maintained for at least 40
days pi. Enriched neuron populations were obtained and individual neurons were
assayed for the presence of the latent viral genome as described [77],[79].

Quantification of viral genomes by real time PCR assay

Isolation and quantification of total DNA from TG and quantification of total
viral genomes by real time PCR was performed as detailed previously [28].

In vivo reactivation

Latent HSV was induced to reactivate in the ganglia of mice in vivo using
hyperthermic stress (HS) and at 22 hours post induction TG were assayed for
infectious virus as detailed previously [5].

In vitro explant reactivation

Latently infected ganglia were aseptically removed and placed into Minimum
Essential Media (MEM) supplemented with 5% newborn calf serum and
incubated at 37°C in a 5% CO2 incubator. At the
indicated times post explant, ganglia were homogenized and assayed for
infectious virus as for reactivation in vivo [24].

Antibodies and immunohistochemistry

Colocalization of b-gal activity and HSV lytic viral proteins was carried out by
first histochemically staining whole TG by incubation in x-gal (Sigma) followed
by paraffin embedding of TG, sectioning and immunohistochemical detection of
viral proteins. Where indicated, HSV proteins were also detected in whole
ganglia using whole ganglia immunohistochemistry (WGIHC). Primary antibodies
utilized include rabbit anti-HSV (AXL237, Accurate), rabbit anti-VP16 antibody
(clonetech), and secondary antibody utilized was HRP labeled goat anti-rabbit
(Vector). These methods and the dilutions and characterizations of antibodies
utilized have been detailed extensively in previous reports [4],[55].

Acknowledgments

We thank David Leib for HSV mutants ΔTfi and ΔTfiR, Peter
O'Hare for helpful discussion, and Steven McKnight for thoughtful comments
on the manuscript. We thank Cheryl Tansky and Tara Riddle for expert technical
assistance.

4.
Sawtell NM (2003) Quantitative Analysis of Herpes Simplex Virus Reactivation In
Vivo Demonstrates that Reactivation in the Nervous System Is Not Inhibited
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